Approximately 62 million Americans have one or more types of cardiovascular disease with coronary heart disease (CHD) and stroke afflicting more than 17 million patients in the United States alone (Ref: American Heart Association. 2002 Heart and Stroke Statistical Update. Dallas, Tex.: American Heart Association; 2001). Despite numerous therapeutic options and technological advances, morbidity and mortality from these diseases are exceedingly high; in fact, cardiovascular diseases are the leading cause of death in the United States claiming 2 of every 5 deaths. Consequently, new approaches to the treatment and prevention of cardiovascular diseases are needed.
The concept that atherosclerosis, the leading cause of CHD, is a process of passive accumulation of lipid in arterial walls eventually leading to the development of symptomatic cardiovascular disease is no longer tenable (Ref: Scientific American 2002 (May): 46-55). Instead, this hypothesis is being replaced by evidence that cardiovascular disease represents a chronic inflammatory process (Ref: Circulation 2002; 105:1135-43). It has been proposed that underlying and preceding acute coronary or cerebrovascular events are “vulnerable” (or high-risk) atherosclerotic plaque(s). Inflammation is thought to be a major contributing factor to plaque instability and rupture leading to unstable angina (UA) and acute myocardial infarction (AMI). Vulnerable plaques have been shown to be frequently present in numerous anatomically distinct locations rather than isolated to a single (culprit) lesion (Ref: Circulation 2003;107:2072-2075). The multifocal nature of vulnerable plaques has been documented in autopsy series and in studies using angiographic, intravascular ultrasound (IVUS), angioscopic, or thermography techniques. Clinical endpoint data (e.g., death, myocardial infarction, stroke) that further support these hypotheses are derived from epidemiological data, retrospective hypothesis-generating analyses of completed clinical trials, and prospective evaluation in clinical trials (summarized below).
Epidemiological data from the Physician's Health Study provides compelling evidence that markers of inflammation (e.g., C-reactive protein [CRP], fibrinogen, interleukin-6, soluble intracellular adhesion molecule-1 [sICAM-1]) are predictive of the future risk of developing AMI (Ref: Circulation 1999;100:1148-1150). Subsequently, more than a dozen population-based epidemiological studies have reported similar observations (Ref: Circulation 2002; 105:1135-43). CRP (and high sensitivity CRP [hs-CRP]) has been the most widely studied inflammatory marker across these epidemiological studies. CRP appears to be an independent predictor of subsequent cardiovascular events (AMI, death) in both primary prevention and secondary prevention patient populations.
Experimental and clinical evidence supports the notion that reduction of inflammation leads to a reduction in clinical events. Aspirin has been shown in the Physician's Health Study to be associated with a reduction of inflammation, as measured by CRP, and is associated with a concomitant reduction of coronary events (Ref: Circulation 1999; 100:1148-1150). To what extent anti-inflammatory effects/CRP reduction vs. antiplatelet effects of aspirin had on this finding in a population of apparently healthy men is not known. Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (“statins”; e.g., pravastatin, simvastatin, atorvastatin, fluvastatin, and lovastatin) serve as a second example of an existing therapy that has anti-inflammatory properties. In addition to their effects on serum lipids, statins also reduce CRP (Ref: Circulation 2002; 105:1135-43). The Pravastatin in the Cholesterol and Recurrent Events (CARE) study provided the first clinical evidence that statin therapy lowers CRP in a fashion unrelated to low-density lipoprotein (LDL) or high-density lipoprotein (HDL) cholesterol. The magnitude of relative risk reduction for subsequent cardiovascular events in this hypercholesterolemic population was greater among subjects who also had evidence of inflammation (i.e., CRP elevation) as compared with those without evidence of inflammation (Ref: Circulation 1999; 100:230-235). This observation was prospectively validated in the Pravastatin Inflammation CRP Evaluation (PRINCE) study and also reported in analysis of several other trials with a variety of statins (Ref: Circulation 2002; 105:1135-43).
Evidence dissociating the benefits of statin therapy in patients with elevations in CRP from those with elevation of cholesterol are derived from the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS; Ref: New Engl J Med 2001;344:1959-1965). This study was a primary prevention study using lovastatin in a population with low to moderate cardiovascular risk. In this study, the magnitude of risk reduction afforded by lovastatin as compared with placebo was nearly as great in the patients with low levels of LDL/high levels of CRP as compared with those with high levels of LDL/low levels of CRP.
The above findings suggest that inflammation represents a novel risk category that is not currently addressed as standard of care in clinical practice guidelines. Since approximately half of all heart attacks occur in populations with normal cholesterol levels, identification and modulation of newly identified risk factors would be an important strategy for reducing cardiovascular morbidity and mortality. The population that fits within this cohort of low-LDL/high-CRP has been estimated to be approximately 25 million Americans (Ref: Circulation 2002; 105:1135-43). The elevation of CRP appears to occur in a graded fashion consistent with subsequent risk of cardiovascular events. Specifically, elevated CRP (>3 mg/dL) is found in 10% of healthy individuals, but is elevated in <20%, >65%, and >90% in patients with chronic stable angina, unstable angina (Braunwald class IIIb), and AMI preceded by USA, respectively (Ref: Circulation 2002; 105:1135-43). Therefore, inflammatory markers represent an opportunity for identification and pharmacological intervention in populations at risk for the development of cardiovascular events.
Other markers of inflammation are emerging that may either replace or augment the utility of CRP measurement. Recently, novel markers of inflammation, including increases in soluble CD40 ligand (sCD40L) and decreases in serum interleukin-10 (IL-10) concentration, have been associated with increased cardiovascular morbidity and mortality. Evidence suggests that CD40L is important in atherosclerotic plaque destabilization. CD40L shed from stimulated lymphocytes is pro-inflammatory causing upregulation of inflammatory cytokines and adhesion molecules. Moreover, CD40L also promotes coagulation by inducing expression of tissue factor in macrophages and endothelial cells and also activates glycoprotein IIb/IIIa (Refs: Proc. Natl. Acad. Sci. 1997;94:1931-1936; Nature 1998;394:200-203; Circulation 2002; 106:896-899).
Epidemiological evidence from the Women's Health Study demonstrated elevated serum sCD40L concentrations are associated with a graded and continuous rise in cardiovascular risk (Ref: Circulation 2001;104:2266-2268). The increase in cardiovascular risk was nearly 12-fold higher among women with the highest sCD40L concentrations. Similar findings were also observed in the c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE) study (Ref: NEJM 2003;348:1104-1111). In this study, a graded and continuous rise in cardiovascular risk (death or nonfatal myocardial infarction) was noted among placebo-treated subjects based upon quintiles of baseline sCD40L. This difference in cardiovascular risk was evident at both early (24 hour) and late (6 month) endpoint determinations. A similar analysis from the CAPTURE trial investigating the anti-inflammatory cytokine IL-10 revealed that placebo-treated patients with high levels of IL-10 (i.e., high anti-inflammatory cytokine levels) had a reduced risk of death (Ref: Circulation 2003; 107:2109-2114). Patients in the highest quartile of serum concentrations of the anti-inflammatory cytokine IL-10 had a >50% reduction in mortality rate as compared with those patients in the lowest quartile of serum IL-10 levels. Moreover, if at the time of hospital discharge patient populations are dichotomized as either high or low IL-10 levels there was an observed adjusted hazard ratio for mortality of 0.38 (i.e., a 62% relative risk reduction) at 6 months favoring subjects with high (anti-inflammatory) IL-10 levels.
In summary, the concept of cardiovascular diseases occurring as a passive process mediated by lipid deposition alone is antiquated. There is a substantial increase in experimental and clinical evidence that suggests cardiovascular disease is a manifestation of a chronic inflammatory process and that intervention in this inflammation may reduce patient morbidity and mortality. Currently, however, the cellular and molecular mediators of these processes are only now being elucidated. Elucidation for these processes is hampered by the lack of an appropriate preclinical model for acute coronary syndrome (ACS). Despite the lack of knowledge of these precise mechanisms, the anti-inflammatory effects of statins appear to validate the concept that pharmacological intervention in patients with elevated markers inflammation leads to reductions of cardiovascular morbidity and mortality. Notably, these benefits are afforded through the serendipitous pleiotropic anti-inflammatory activities of statins. Further understanding of the cellular and molecular processes may lead to the development of specific and more efficacious anti-inflammatory agents to further reduce cardiovascular morbidity and mortality among these patients.
In general, the magnitude of the T-cell response is determined by the co-stimulatory response elicited by the interaction between T-cell surface molecules and their ligands (Mueller, et al., 1989 Ann. Rev. Immunol. 7:445-480). Key co-stimulatory signals are provided by the interaction between T-cell surface receptors, CD28 and CTLA4, and their ligands, such as B7-related molecules CD80 (i.e., B7-1) and CD86 (i.e., B7-2), on antigen presenting cells (Linsley, P. and Ledbetter, J. 1993 Ann. Rev. Immunol. 11: 191-212). T-cell activation in the absence of co-stimulation results in anergic T-cell response (Schwartz, R. H., 1992 Cell 71:1065-1068) wherein the immune system becomes nonresponsive to stimulation.
Soluble forms of CD28 and CTLA4 have been constructed by fusing variable (V)-like extracellular domains of CD28 and CTLA4 to immunoglobulin (Ig) constant domains resulting in CD28Ig and CTLA4Ig. A nucleotide and amino acid sequence of CTLA4Ig is shown in FIG. 24 with the protein beginning with methionine at position +1 or alanine at position −1 and ending with lysine at position +357. CTLA4Ig binds both CD80-positive and CD86-positive cells more strongly than CD28Ig (Linsley, P., et al., 1994 Immunity 1:793-80). Many T-cell-dependent immune responses have been found to be blocked by CTLA4Ig both in vitro and in vivo. (Linsley, P., et al., 1991b, supra; Linsley, P., et al., 1992a Science 257:792-795; Linsley, P., et al., 1992b J. Exp. Med. 176:1595-1604; Lenschow, D. J., et al. 1992 Science 257:789-792; Tan, P., et al., 1992 J. Exp. Med. 177:165-173; Turka, L. A., 1992 Proc. Natl. Acad. Sci. USA 89:11102-11105).
To alter binding affinity to natural ligands, such as B7, soluble CTLA4Ig fusion molecules were modified by mutation of amino acids in the CTLA4 portion of the molecules. Regions of CTLA4 that, when mutated, alter the binding affinity or avidity for B7 ligands include the complementarity determining region 1 (CDR-1 as described in U.S. Pat. Nos. 6,090,914, 5,773,253, 5,844,095; in copending U.S. patent application Ser. No. 60/214,065; and by Peach et al, 1994. J. Exp. Med., 180:2049-2058) and complementarity determining region 3 (CDR-3)-like regions (CDR-3 is the conserved region of the CTLA4 extracellular domain as described in U.S. Pat. Nos. 6,090,914, 5,773,253 and 5,844,095; in copending U.S. patent application Ser. No. 60/214,065; and by Peach, R. J., et al J Exp Med 1994 180:2049-2058; the CDR-3-like region encompasses the CDR-3 region and extends, by several amino acids, upstream and/or downstream of the CDR-3 motif). The CDR-3-like region includes a hexapeptide motif MYPPPY (SEQ ID NO.: 20) that is highly conserved in all CD28 and CTLA4 family members. Alanine scanning mutagenesis through the hexapeptide motif in CTLA4, and at selected residues in CD28Ig, reduced or abolished binding to CD80 (Peach, R. J., et al J Exp Med 1994 180:2049-2058; U.S. Pat. No. 5,434,131; U.S. Pat. No. 6,090,914; U.S. Pat. No. 5,773,253.
Further modifications were made to soluble CTLA4Ig molecules by interchanging homologous regions of CTLA4 and CD28. These chimeric CTLA4/CD28 homologue mutant molecules identified the MYPPPY (SEQ. ID NO:20) hexapeptide motif common to CTLA4 and CD28, as well as certain non-conserved amino acid residues in the CDR-1- and CDR-3-like regions of CTLA4, as regions responsible for increasing the binding avidity of CTLA4 with CD80 (Peach, R. J., et al., 1994 J Exp Med 180:2049-2058).
Soluble CTLA4 molecules, such as CTLA4Ig, CTLA4 mutant molecules or chimeric CTLA4/CD28 homologue mutants as described supra, introduce a new group of therapeutic drugs to treat cardiovascular diseases.